Her2 expression and gene amplification is rarely detectable in patients with oral squamous cell carcinomas.

PURPOSE: Her2 (ErbB2) transforms cells when overexpressed and is an important therapeutic target in breast cancer. Contrary to breast cancer, studies on Her2 overexpression and gene amplification in squamous cell carcinomas of the head and neck region described largely different results. This study was undertaken to learn more on the prevalence and clinical significance of HER2 amplification and overexpression in squamous cell carcinomas of the head and neck. MATERIALS AND METHODS: Her2 expression and gene amplification was analyzed by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) on two tissue microarrays composed of 427 squamous cell carcinomas of the head and neck region and 222 oral squamous cell carcinomas. Results were compared with clinicopathological features. RESULTS: Her2 expression and gene amplification was rarely detectable in squamous cell carcinomas of the head and neck region and unrelated to tumor phenotype or survival of the patients with oral squamous carcinoma. DISCUSSION: Our results demonstrate that Her2 protein and gene amplification was only detectable in a small subset of squamous cell carcinomas of the head and neck region as well as oral squamous cell carcinomas. However, it can be speculated that those few patients with Her2 overexpressing and gene amplificated tumors may possibly benefit from an anti-Her2 therapy.

Parapharyngeal liposarcoma: a case report.

BACKGROUND: Parapharyngeal liposarcoma is a very rare malignant tumor that often causes nonspecific clinical symptoms, such as progressive dysphagia, globus sensation and/or respiratory disturbances. The combination of radiological imaging techniques and histopathological analysis provides information for diagnosis; however, the pathogenesis is still uncertain. CASE PRESENTATION: A 30-year-old male patient presented with a pharyngeal cavity mass, which had been present for 2 years. The clinical syndrome included obstructive sleep apnea symptoms (i.e., respiratory disturbances, excessive daytime somnolence, and headache) and difficulty swallowing. The radiological examination (CT) demonstrated that there was a low-density irregular solid lesion on the posterior wall of the oropharynx and laryngopharynx, which descended to the superior mediastinum and extended to the left parapharyngeal space and sternocleidomastoid muscle. The boundaries of the lesion were clear, and the lesion's density was nonuniform. Several septations inside the lesion were observed. The CT values of the lesion at the epiglottis and the vocal folds were 11 HU and minus 30 HU, respectively. After enhanced scanning, there was no apparent enhancement of the lesion: the surrounding tissue and blood vessels were squeezed and shifted, but the neighboring sclerotin of the cervical vertebrae was not invaded. The mass was removed via a transcervical approach, resulting in a complete amelioration of the patient's symptoms. Interestingly, immunohistochemistry showed that the tumor cells expressed members of the B7 superfamily, including B7-H1, B7-DC and B7-H3. In addition, the expression of TIM-containing molecules, including TIM-3 and TIM-4, was observed. CONCLUSIONS: CT and MRI demonstrated that the mass was a parapharyngeal liposarcoma. Furthermore, carcinoma-associated B7 and TIM-containing molecules were observed in the tissue, indicating that these molecules are most likely active in the pathogenesis of this disease. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1907794973876202.

Intratumoral lymphatic vessels and VEGF-C expression are predictive factors of lymph node relapse in T1-T4 N0 laryngopharyngeal squamous cell carcinoma.

BACKGROUND: The presence of intratumoral lymphatic vessels (ILVs) and the expression of vascular endothelial growth factor-C (VEGF-C) in tumour cells have been studied as markers of lymphangiogenesis in order to evaluate their role in metastatic dissemination in laryngopharyngeal squamous cell carcinoma. METHODS: A retrospective study was performed in 76 patients of N0 laryngopharyngeal carcinoma. with variable tumour size (T1-T4), histological grade, and location (supraglottic, glottic and hypopharyngeal). The presence of ILVs, as revealed by the expression of PA2.26 antigen and VEGF-C expression, were determined by immunohistochemistry (IHC). Low-grade and high-grade lymphangiogenesis were defined by qualitative and quantitative criteria. RESULTS: Multivariate analysis revealed low-grade ILV and VEGF-C expression to be associated respectively with 30.3- and 16.2-fold higher probabilities of cervical lymph node relapse (P = 0.005 and P = 0.032) and with 16.2- and 8.44-fold shorter disease-free survival (P = 0.009 and P = 0.045). CONCLUSIONS: Low-grade ILV and VEGF-C expression are independent predictive factors of cervical lymph node relapse and shortening of time to relapse in N0 laryngopharyngeal carcinoma.

Pharyngeal teratocarcinosarcoma: review of the literature and report of two cases.

Teratocarcinosarcomas are rare malignant neoplasms histologically characterized by the presence of benign and malignant epithelial and mesenchymal elements. They are seen almost exclusively in the sinonasal tract of men. We report two cases of teratocarcinosarcomas involving the posterior pharyngeal wall in a 55-year-old male and 60-year-old men. The tumors consisted of epithelial components including squamous, neuroendocrine, and glandular structures; neuroepithelium, and mesenchymal components with prominent rhabdomyoblastic, osteoblastic and chondroid differentiation. Immunohistochemical studies demonstrated markers characteristic of each component. The tumors were resected, and the patients received postoperative radiation therapy. One patient is alive with recurrent tumor 33 months after treatment and the other died 26 months after radiation therapy with distant metastasis.

Follicular dendritic cell sarcoma of the pharyngeal region: histologic, cytologic, immunohistochemical, and ultrastructural study of three cases.

Follicular dendritic cell sarcoma is a tumor of recent description and characterization; it is often underdiagnosed because it is easily confused with other entities. Three cases of follicular dendritic cell sarcoma are described in the present article. The first occurred in the parapharyngeal space in a 29-year-old woman who developed multiple recurrences over the span of 10 years. The second was located in the left tonsil in a 48-year-old man, and the third case developed in the parapharyngeal space in a 26-year-old man. All cases were positive for CD21 and CD35 and ultrastructurally they displayed a morphologic spectrum. The first case featured spindle cells with interdigitated long cell processes joined by well-developed desmosomes. In the other two cases there were round to ovoid cells with interwoven processes connected by occasional desmosomes. Including these three cases, a total of 20 follicular dendritic cell sarcoma of the pharyngeal region have been reported to date. The clinical behavior of these tumors is similar to other low-grade sarcomas.

P105 as a prognostic indicator in patients irradiated for locally advanced head-and-neck cancer: a clinical/laboratory correlative analysis of RTOG-9003.

PURPOSE: In a previous retrospective study, p105 AD, a proliferation-associated nuclear antigen density (AD), was found to be an independent prognostic factor for patients irradiated for locally advanced head-and-neck cancer. We sought to confirm this finding by analyzing patients entered on RTOG 9003, a Phase III randomized trial of altered fractionation radiotherapy. METHODS AND MATERIALS: Paraffin blocks of pretreatment biopsies of the primary tumor of patients with Stage III or IV squamous cell carcinoma of the oral cavity, oropharynx, or supraglottic larynx, or Stage II squamous cell carcinoma of the hypopharynx or base of tongue entered on RTOG 9003 were prospectively collected at patient entry. From these paraffin blocks, areas of tumor were selected based on histologic examinations and sectioned. Nuclear suspensions were then prepared and processed for p105 antibody and DNA staining. Flow cytometric quantification of p105 labeling indices and DNA content were then performed for correlation with local-regional control and survival. RESULTS: Paraffin blocks of tumor biopsies from 457 of 1073 patients entered were available for p105 determination. There was no significant difference in pretreatment characteristics between patients who had paraffin blocks available or not available. The median (range) of p105 labeling index (LI-C), p105 labeling index of cells in S phase (p105 LI-S), and p105 AD were 56 (range: 6-99), 8.255 (range: 0.913-23), and 67 (range: 5-364), respectively. Multivariate analysis of prognostic factors showed that T stage, N stage, Karnofsky performance status, and fractionation schedule were significant for local-regional control (p < 0.0001, 0.0011, <0.0001, and 0.007, respectively) and T stage, N stage, Karnofsky performance status, and tumor grade were significant for survival (p = 0.018, 0.002, <0.0001, and 0.0058, respectively). Neither p105 LI-C nor p105 LI-S nor p105 AD nor DNA ploidy was significant for local-regional control or survival. CONCLUSION: p105 labeling indices, antigen density, and DNA ploidy do not predict the outcome of patients irradiated for advanced squamous cell carcinomas of the head and neck.

Extracellular matrix-regulated p53 expression and nuclear localization in cultured Detroit 562 cells derived from pharyngeal carcinoma.

Tumor suppressor p53 functions as a transcriptional factor that regulates the cell cycle and apoptosis. A mutated p53 gene can result in decreased sequence-specific DNA binding and transcriptional activity of the p53 protein. In this study, we examined the regulatory role of the extracellular matrix components in p53 expression and nuclear localization in a Detroit 562 cell line derived from a pharyngeal carcinoma. When cultured on a polystyrene surface, type I collagen gel, or Matrigel containing basement membrane components, Detroit 562 cells showed a distinct response to extracellular matrix components morphologically. As shown by Western blotting, Detroit 562 cells cultured on Matrigel displayed an increased expression of p53 protein as well as an elevated nuclear p53 level, as compared with the cells cultured on the polystyrene surface or type I collagen gel. When cultured on Matrigel, nuclear p53-positive cells were exclusively localized to the outer surface layer of the cell clusters, whereas most of the inner cells showed no p53 expression. In Detroit 562 cell clusters on Matrigel, proliferative activities, as evaluated by proliferationcell nuclear antigen staining and bromo-deoxyuridine incorporation assay, were evenly distributed; virtually no apoptotic cells, as evaluated by the fluorescence TUNEL assay, were detected in the cell clusters, suggesting that the peculiar localization of nuclear p53-positive cells was not directly related to cell proliferation or apoptosis. These results indicate that p53 expression and its localization in Detroit cells were modulated by extracellular matrix signals, particularly by the basement membrane components in Matrigel.

Extranodal follicular dendritic cell sarcoma of the head and neck region: three new cases, with a review of the literature.

Extranodal follicular dendritic cell (FDC) sarcoma of the head and neck region is uncommon, with 16 well-documented cases previously reported (four in the tonsil, four in the pharynx, two in the palate, five in the soft tissue, and one in the thyroid). We here report an additional three cases of extranodal FDC sarcoma in the tonsil (two cases) and pharynx (one case). In these new cases, the neoplastic cells were arranged in diffuse, fascicular, and vaguely whorled growth patterns. A background lymphocytic infiltrate was sprinkled throughout the neoplasms, with focal prominent perivascular cuffing. Scattered multinucleated giant cells were present. Immunohistochemically, tumor cells were strongly and diffusely positive for follicular dendritic cell markers CD21 and CD35. Tumor cells were diffusely positive for fascin and negative for leukocyte common antigen, S-100 protein, cytokeratin, and Epstein-Barr virus (EBV) latent membrane protein-1 (EBV-LMP). EBV was also not detected in the tumor cells by in situ hybridization for EBV-encoded RNAs. FDC sarcomas are probably an underrecognized neoplasm, especially when they occur in extranodal sites in the head and neck region. Two of the three new cases we report were initially misdiagnosed, and five cases of extranodal FDC sarcoma in the head and neck region reported in the recent literature were initially misdiagnosed. Our aim is to complement the current understanding of this neoplasm and alert pathologists to this rare entity in this region to avoid misdiagnosis. Recognition of extranodal FDC sarcoma requires a high index of suspicion, but this tumor has numerous distinctive histological features that should bring the neoplasm into the differential diagnosis. Confirmatory immunohistochemical staining with follicular dendritic cell markers such as CD21 and/or CD35 is essential for the diagnosis. Correct characterization of this neoplasm is imperative given its potential for recurrence and metastasis.

Expression of granulocyte colony-stimulating factor receptor and platelet-derived endothelial cell growth factor in oral and oropharyngeal precancerous lesions.

Epithelial hyperplasia and dysplasia have been diagnosed as precancerous lesions and have been discussed in relationship to carcinogenesis. We analyzed the immunohistochemical expression of granulocyte colony-stimulating factor receptor (G-CSFR) and platelet-derived endothelial cell growth factor (PD-ECGF) in oral and oropharynx; 33 samples of normal epithelium, 28 samples of hyperplasia, 16 samples of dysplasia and 58 samples of squamous cell carcinoma. Also, we examined mean vessel density (MVD) by using CD34 staining and proliferating cell nuclear antigen (PCNA) staining. Dysplasia and head and neck Squamous Cell Carcinoma (SCC) exhibited higher G-CSFR expression and MVD than normal or hyperplastic epithelium (p <0.01). In the PD-ECGF staining, significant differences were found between SCC and normal epithelium, hyperplasia and dysplasia (p<0.001). In dysplasia and hyperplasia, PD-ECGF expression was significantly correlated with PCNA expression (r=0.345, p=0.025), however it was not correlated with the MVD. G-CSFR expression was not correlated with either PCNA or MVD. These results suggest that G-CSFR and PD-ECGF might be concerned with different carcinogenesis pathways of the squamous cells in the oral region and that PD-ECGF may be concerned with epithelial proliferation rather than angiogenesis.

Clinical trial with escalating doses of the antiepidermal growth factor receptor humanized monoclonal antibody EMD 72 000 in patients with advanced squamous cell carcinoma of the larynx and hypopharynx.

In this open uncontrolled phase I study, nine patients with stage III and IV squamous cell carcinoma of the head and neck (SCCHN) were treated with five administrations of the humanized antiepidermal growth factor receptor monoclonal antibody EMD 72000 in three consecutive ascending dose groups. Loading doses of 100 mg (group I), 200 mg (group II), and 400 mg (group III) were followed by four weekly maintenance doses of half the loading doses, i.e. 50, 100, and 200 mg, respectively. Two EMD 72000 administrations were scheduled before and three after surgery. The objectives of this trial were (a) to investigate the safety and toxicity of multiple EMD 72000 doses, (b) to determine the cumulative maximum tolerated dose of EMD 72000 at dosages between 300 mg and 1,200 mg, and (c) to determine the serum pharmacokinetics of EMD 72000. In total, 102 adverse events (AEs) were reported: five of toxicity grade 3, 18 of toxicity grade 2, 66 of toxicity grade 1, and 38 of toxicity grade 0. All AEs of toxicity grade 3 were considered to be not or remotely related to EMD 72000. The most frequent study drug-related AEs were fever and a transient elevation of liver enzymes. In all patients, the time to reach peak serum concentrations (tmax) was within 1-3 h of the start of each EMD 72000 infusion. Average peak serum concentrations (Cmax) after correction for dosage appeared to be dose-independent, whereas the half-life (t1/2) showed dose dependency. In conclusion, EMD 72000 was very well tolerated in patients with advanced stage SCCHN. The pharmacokinetic data from this trial suggest the feasibility of conducting future studies with weekly doses of 200 mg EMD 72000.

Multiparameter flow cytometry for simultaneous assessment of p53 protein expression and cellular DNA content in oral squamous cell carcinomas: evidence for the development of aneuploid clones from p53-deficient diploid progenitor cells.

Diploid tumour cells regularly continue to progress after the development of aneuploid cell populations in head and neck squamous cell carcinomas. The coexistence of aneuploid clones with their diploid progenitor cells provides a unique opportunity to study the order of appearance of p53 mutation and aneuploidy in the same tumour. Multiparameter flow cytometry was therefore applied to 22 oral squamous cell carcinomas to simultaneously assess cellular DNA content and p53 protein expression on a single-cell basis. Concurrent measurements of cytokeratin expression served to identify tumour cells of epithelial origin. One of 5 diploid and 2 of 17 aneuploid carcinomas were p53-negative. For 15 p53-positive aneuploid tumours, overexpression of p53 protein was identified for the aneuploid clones as well as for coexisting diploid tumour cell populations in 14 cases. On the understanding that coexisting diploid and aneuploid tumour cell populations have a common clonal origin, these results provide evidence that aneuploid tumour clones typically develop from p53-deficient diploid progenitor cells. Loss of wild-type p53 function may therefore contribute to the development of aneuploidy in head and neck cancer.

Expression of matrix metalloproteinases and tissue inhibitor of metalloproteinases in laryngeal and pharyngeal squamous cell carcinoma: A quantitative analysis.

Squamous cell carcinomas of the head and neck are known for their aggressive growth and propensity to metastasize. Invasion is facilitated by matrix metalloproteineases (MMPs). Tissue inhibitors of MMPs (TIMPs) negatively regulate MMP activity. MMP and TIMP expression in head and neck squamous cell carcinomas was determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). qRT-PCR allows measurement of several mRNAs from as little as 4 microg of total cellular RNA. We measured MMP-1, MMP-2, MMP-9, and TIMP-1 expression in 8 specimens of primary tumors and adjacent normal tissue. MMP-1 was overexpressed in 6 of 8 tumors, and MMP-9 was overexpressed in 4 of 7 tumors. MMP-2 was expressed in 3 of 8 tumors and 3 of 8 normal samples. TIMP-1 was expressed in all specimens. This work demonstrates that qRT-PCR can be used to examine expression of specific mRNAs in clinical specimens. Therefore this method provides another tool for the molecular analysis of tumors.

Predictive value of cathepsin-D for cervical lymph node metastasis in head and neck squamous cell carcinoma.

BACKGROUND: Prognosis of head and neck squamous cell carcinoma (HNSCC) is strongly associated with cervical lymph node metastasis. Cathepsin-D is a lysosomal protease expressed in all cells. Its role in extracellular matrix degradation is postulated to promote tumor invasion and metastasis. Increased cathepsin-D has been demonstrated in cervical lymph node metastasis in HNSCC. METHODS: Formalin fixed tumor biopsy samples from 34 patients with HNSCC of the oral cavity, oropharynx, or hypopharynx were analyzed for the presence of cathepsin-D by immunohistochemistry (1:8000, Calbiochem, Cambridge, MA). Tumors were considered positive if >50% of cells showed strong cytoplasmic staining. RESULTS: All patients had T1 or T2 lesions ranging in size from 1-4 cm and 19 (56%) had cervical metastasis. Eight (24%) were well differentiated and 26 (76%) were moderately or poorly differentiated. Thirteen tumors (38%) had high cathepsin-D expression that was strongly associated with cervical lymph node metastasis (p = 0.008). When adjusted for tumor stage and grade, cathepsin-D positivity was nearly twice as likely to be associated with node metastasis (p = 0.011). CONCLUSIONS: We demonstrated cathepsin-D expression in biopsies from a subset of patients with HNSCC and a strong association between this protease and cervical lymph node metastases. Cathepsin-D is a potential independent predictor of cervical lymph node metastasis in HNSCC and merits additional study.

Cytokeratin alterations as diagnostic and prognostic markers of oral and pharyngeal carcinomas. A prospective study.

Cytokeratin (CK) alterations have been reported in carcinomas from different anatomical sites, and these have been associated with specific aspects of tumour behaviour. In order to assess the relationships between CK modifications and future tumour behaviour, we conducted the present prospective study on 26 squamous cell carcinomas (SCC) of oral and pharyngeal mucosae and corresponding controls. Cytokeratins were investigated using two-dimensional gel electrophoresis and immunofluorescence techniques. All healthy tissues, oral lining and oropharyngeal mucosae, expressed the oesophageal type CKs, including CK 19. Other simple epithelial CKs (7, 8, 17 and 18) were not detected. In carcinomas originating from corresponding sites, expression of oesophageal CKs varied widely from one specimen to another, and simple epithelial keratins were often found. Statistical analysis indicated correlations between CK expression and the clinicopathological data of SCC patients. Small tumour size was strongly associated with the expression of CKs 10 and 19. Interestingly, an absence of lymph node involvement was significantly associated with CK 18 expression. Tumours giving rise to recurrences, metachronous tumours, and distant metastasis were significantly associated with an absence of CK 13. These results suggest that CKs 10, 19, 18 and 13 could be reliable diagnostic and prognostic markers in the assessment of oral and pharyngeal squamous carcinomas.

Prognostic value of histological and biological markers in pharyngeal squamous cell carcinoma: a case-control study.

Between 1980 and 1985, 914 patients with head and neck squamous cell carcinoma underwent lymph node dissection in our institution. The prognostic value of clinical factors has already been reported (Mamelle et al, 1994, Am J Surg 168: 494-498). We present here a comparison of biological characteristics of pharyngeal tumours in patients who developed distant metastasis and in patients without metastasis, matched on tumour site, node site and size, and year of diagnosis. Tumour differentiation, keratinization, vascular emboli, immunohistochemical expression of p53, c-erb-B2, Rb and bcl2 were first assessed in 31 pairs of patients. Factors of potential interest were then determined in 32 additional pairs of patients. Statistical analysis showed that the risk of distant metastasis was halved in patients with tumours expressing c-erb-B2 compared with patients with c-erb-B2-negative tumours (P = 0.05). The significance of c-erb-B2 expression and its potential value as a prognostic factor is discussed.

Schwannomas of the sinonasal tract and nasopharynx.

Approximately 45% of benign peripheral nerve sheath tumors occur in the head and neck region. Of these, schwannomas (neurilemomas) arising from the nasal cavity and paranasal sinuses account for less than 4%. Pathologic features of this subset are not well documented. We report a series of five cases of sinonasal schwannoma and one in the nasopharynx. The male-to-female ratio was equal, and the age at presentation ranged from 38 to 65 years of age (median, 52 yr). Four of the lesions were located within the nasal cavity, one arose from the maxillary sinus, and one originated in the nasopharynx, with extension into the Eustachian tube. Two cases showed local bony destruction, with intracranial extension. Presenting clinical symptoms included nasal obstruction, epistaxis, rhinorrhea, anosmia, facial swelling, headache, and serous otitis media; the two cases with intracranial spread also presented with visual disturbances. All of the six cases were treated by surgical excision. Clinical follow-up in five cases ranged from 6 to 48 months (median, 27 mo). Histologically, all of the lesions shared many cytomorphologic features common to schwannomas arising at other sites, and all of the six cases showed strong, diffuse immunoreactivity for S-100 protein. Four cases showed features of the cellular variant, and one showed focal granular cell change. An unusual and previously poorly documented histologic feature, distinct from schwannomas arising at most other anatomic sites, was a lack of encapsulation, which, when combined with hypercellularity, often raised suspicion of malignancy. Because none of the cases in this series has shown either local recurrence or postoperative metastasis to date, lack of encapsulation and locally destructive growth in an otherwise histologically typical schwannoma arising at this site should not suggest malignant potential.

[Biological behavior of hypopharyngeal carcinoma].

Hypopharyngeal squamous cell carcinomas (HPC) has an extremely poor prognosis. Characteristics of cell lines of head and neck squamous cell carcinomas including HPC were studied by various methods, e.g., chemosensitivity test and the immunohistochemistry staining method, to determine whether this poor prognosis is due to the biological behavior of this cancer. An HPC cell line was found to be resistant to anti tumor drugs, i.e., PEP, MTX and CPM and moderately sensitive to CDDP, 5-FU and ADM. Thermoresistance to hyperthermatic treatment and weak expression of ICAM-1 on the HPC cell line were observed. DNA synthesis by the HPC cell line was induced by stimulation with a low concentration of EGF and the amount of EGFR on these HPC cells was very high. In addition, cyclinD1 overexpression was found in the HPC cell line. Based on the above findings, further analysis of hypopharyngeal carcinoma cells and the development of a new treatment modality to control tumor growth and metastatic factors influencing the poor outcome are necessary to improve the prognosis of this cancer.

Detection of nodal micrometastases in head and neck cancer by serial sectioning and immunostaining.

We investigated the incidence of micrometastases from squamous cell carcinomas of the head and neck in neck dissection specimens originally staged as pNO. A total of 76 dissection specimens from 60 patients were evaluated using serial microscopic sectioning in 10-microns intervals, H & E staining, and immunostaining with an antibody to pan-cytokeratin. Examination of 1,020 lymph nodes from 76 neck dissection specimens revealed 8 micrometastases (7.9%) in 6 specimens from 6 patients with oral and pharyngeal primaries, resulting in upstaging. Six micrometastases were located in lymph nodes 3 to 6 mm in diameter. The surgeon should be aware of the relatively high incidence of micrometastases from oral and pharyngeal carcinomas, which are undetectable preoperatively or by routine histopathologic examination. Primary tumor site (oral cavity and pharynx) and certain features of the primary can delineate a group of patients with a higher risk of harboring occult metastases who may benefit from elective treatment of the neck.

Abnormalities and the implication of retinoblastoma locus and its protein product in head and neck cancers.

Abnormalities in the Retinoblastoma tumor suppressor gene (Rb) have been observed in a large number of human cancers. Loss of heterozygosity (LOH) is a common mode of allelic inactivation of Rb and other tumor suppressor genes. We investigated DNA from 45 primary human head and neck cancers to determine LOH at the Rb locus using a polymerase chain reaction-based restriction fragment length polymorphism assay. Of informative cases, we found LOH in 4 of 28 (14%) head and neck cancers. Of immunohistochemical staining of Rb protein, we found that in one of ten LOH negative cases the nuclei of fibroblasts were stained with anti-Rb antibody but there was no nuclear staining tumor cells. These results suggest that inactivation of Rb protein is involved in the carcinogenesis of head and neck cancer at all levels of the process of protein expression: DNA, mRNA and protein.